ABSTRACT
BACKGROUND: The goal of this study was to evaluate the accuracy and interchangeability between continuous cardiac output (CO) measured by electrical velocimetry (COEv) and continuous cardiac output obtained using the pulmonary thermodilution method (COPAC) during living donor liver transplantation (LDLT). METHOD: Twenty-three patients were enrolled in this prospective observational study. CO was recorded by both two methods and compared at nine specific time points. The data were analyzed using correlation coefficients, Bland-Altman analysis for the percentage errors, and the concordance rate for trend analysis using a four-quadrant plot. RESULTS: In total, 207 paired datasets were recorded during LDLT. CO data were in the range of 2.8-12.7 L/min measured by PAC and 3.4-14.9 L/min derived from the EV machine. The correction coefficient between COPAC and COEv was 0.415 with p < 0.01. The 95% limitation agreement was - 5.9 to 3.4 L/min and the percentage error was 60%. The concordance rate was 56.5%. CONCLUSIONS: The Aesculon™ monitor is not yet interchangeable with continuous thermodilution CO monitoring during LDLT. TRIAL REGISTRATION: The study was approved by the Institutional Review Board of Chang Gung Medical Foundation in Taiwan (registration number: 201600264B0 ).
Subject(s)
Cardiac Output/physiology , Liver Transplantation/methods , Lung/physiology , Monitoring, Intraoperative/methods , Rheology/methods , Adult , Aged , Female , Humans , Liver Transplantation/adverse effects , Liver Transplantation/standards , Male , Middle Aged , Monitoring, Intraoperative/standards , Prospective Studies , Rheology/standards , Thermodilution/methods , Thermodilution/standardsABSTRACT
Carboxypeptidase B is a metalloenzyme, which is widely used for commercial and research purposes. Commercially available CPB purified from porcine or bovine pancreas is very expensive, and is not totally free from other proteases. In order to express the rat proCPB in Pichia pastoris, total RNA extracted from SD rat pancreas cells was reversely transcripted to synthesize cDNA, and the proCPB ORF was synthesized by PCR. After digestion with Xho I and EcoR I , the fragment was inserted into pPIC9, and the recombinant plasmid was named as pPIC9-proCPB. By digestion with Sac I , the lined pPIC9-proCPB was transformed into Pichia pastoris strains GS115 with PEG1000 and integrated into their genomes. In the inducement of methanol, recombinant proCPB was successfully expressed in Pichia pastoris, and could be secreted into the supernatant in the culture. After optimizing the fermentation conditions, a higher production could be obtained when GS115-proCPB was induced in BMGY (pH6.0) at 28CC, with addition of 0.5% casein. The yield of recombinant protein reached 500mg/L, achieving over 94% of total protein in the culture supernatant. The purity of recombinant CPB can reach 96% after two step phenyl sepharose F F purification, and 38% of total protein can obtained after optimizing the pufication method. Comparing to the specific activity 180u/mg of CPB purchased from Sigma, the specific activity of recombinant CPB is 110u/mg. Mass spectrometry analyses showed the mass of the recombinant CPB was 35.1 kD, which is very close to the theory value 35.2 kD. Amino acid sequencing of N-terminal of recombinant CPB further indicated proCPB was expressed successfully and modificated correctly after translation.
Subject(s)
Carboxypeptidase B/metabolism , Pichia/genetics , Recombinant Proteins/metabolism , Animals , Carboxypeptidase B/genetics , Carboxypeptidase B/isolation & purification , Catalysis , Cattle , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Enzymologic , Kinetics , Mass Spectrometry , Molecular Weight , Rats , Rats, Sprague-Dawley , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sequence Analysis, Protein , Substrate Specificity , SwineABSTRACT
The fused gene (PTH-TFN) of parathyroid hormone (PTH) gene and transferring N-terminal half-molecule (TFN) gene was amplified by multiple PCR and inserted into pPIC9 vector. The recombinant plasmid pPIC9-PTH-TFN was transformed into Pichia pastoris GS115 by PEG. After methanol induction, the target protein was expressed in fermentation supernatant at high level. The fused protein PTH-TFN with purity being higher than 95% was finally obtained after purification through two-step chromatography: SP Sepharose Fast Flow and Phenyl Sepharose Fast Flow. Western blot analysis and adenylate cyclase assay proved that the fused protein exhibited the bioactivity to stimulate cAMP synthesis and the ability to bind Fe3+ in the Fe3+ saturation study as the recombinant TFN did indicating that TFN could be used as the transcellar carrier of PTH.